PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disorder in which pathogenic T-cells destroy the insulin producing ?- cells of the pancreas. Despite the NOD mouse contributing to our understanding of T1D pathogenesis, it has not proved ideal for testing potentially clinically applicable disease therapies. This lack of clinical translation argues for the need for new mouse models that will provide better pipelines for therapy development. Since the major histocompatibility complex (MHC in mice, HLA in humans) genes are the most potent contributors to T1D susceptibility, the on going creation of HLA-?humanized? NOD mice represents the next level of models to develop possible disease interventions. The Serreze lab has developed several such mice carrying different human HLA class I genes linked to diabetes susceptibility: NOD.?2m-/-.HHD (HLA-A2.1), NOD.?2m-/-.B39 (HLA-B39) and NOD.?2m-/-.A24 (HLA-A24). In order to prevent expression of murine MHC I molecules, these mice carry the ?2m-/- mutation. However, since ?2m is a critical component of the FcRn complex and IgG salvage pathways, these mice are limited for testing antibody-based therapies. Therefore, we have used CRISPR/Cas9 technology to directly eliminate expression of murine MHC class I molecules in NOD mice (designated NOD.MHCI-/-). The transgenes described above encoding human diabetogenic class I transgenes have been crossed into the NOD.MHCI-/- stock. The overall hypothesis of this proposal is that such largely already available HLA-humanized NOD mice can be utilized to develop potentially clinically translatable means to attenuate autoreactive CD8+ T-cell populations of pathogenic significance to a sizeable proportion of T1D patients. Taking advantage of the mouse genetics training I am receiving at The Jackson Laboratory, in Aim 1 I propose utilizing a CRISPR/Cas9-mediated approach to also knock out the sole NOD MHC II (H2-Ag7) variant in the recently developed NOD.MHCI-/- mice. Any combination of HLA-A2.1, B39, A24 class I or DQ8 or DR4 class II transgenes already available on the NOD background can then be crossed into the NOD.MHC-/- stock to generate additional new models for HLA-specific therapy development. Additionally, I propose to test two potential therapies in newly developed NOD.MHCI-/-.A2.1 mice. Aim 2 will test the hypothesis that tolerogenic delivery of HLA-A2.1 restricted peptides (INS1B:5-14, INS1/2A:2-10, IGRP228-236, and IGRP265-273) via PLGA- microspheres can attenuate diabetogenic CD8+ T cell responses directed against these epitopes. In Aim 3 I will test the hypothesis that induction through an irradiation free pre-conditioning regimen of mixed chimerism with MHC-mismatched donor cells can eliminate or inactivate diabetogenic CD8+ T-cells with cross-reactivity to the donor-MHC. Together, this proposal aims to improve current NOD mouse models to develop and validate curative therapies for T1D, while providing me with the necessary training in mouse genetic and genomic editing techniques to eventual create humanized mouse models for other autoimmune disorders.